Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

For experiments using bacteria-infected macrophages, mid-log phase 10 mL cultures of Mtb H37Rv, CDC1551, or HN878, or cultures of E. coli, S. aureus, P. aeruginosa, S. pneumonia, K. pneumonia were pelleted by centrifugation at 3000 g for 10 min at 4°C, and resulting bacterial pellets were suspended in 10 mL of RPMI 1640 / 10% FBS without penicillin and streptomycin, de-clumped using a brief sonication step, and passed 10 times through syringe fitted with 27-gauge needle (VWR, Norm-Ject, USA). Mtb suspensions were then mixed with an additional 10 mL of antibiotic-free RPMI 1640 / 10% FBS, and 0.1 mL aliquots of suspensions were added to T175 flasks containing ~2.5 x 10 7 differentiated THP-1 macrophages cultured in 20 mL antibiotic-free RPMI 1640/10% FBS to obtain a multiplicity of infection (MOI) of 10. After 4 h incubation, cell cultures were washed 3× with 37°C PBS to remove extracellular Mtb bacilli and cultured in RPMI 1640 without FBS for 48h, after which culture supernatant was passed through 0.22um filters to remove Mtb bacilli and generate samples for EV and soluble protein analyses. Culture filtrates were stored at -80 °C while aliquots were inoculated into mycobacterial growth indicator tubes and assessed for Mtb growth after 3-4 weeks of culture to confirm the absence of viable Mtb bacilli remained in these samples. Cultured macrophages were recovered by trypsin digestion and split into samples that were analyzed for viability and employed to generate cell lysates for Western blot analysis of target proteins.
For experiments using culture filtrate protein (CFP), aliquots containing 100 μg CFP were added to T175 flasks containing ~2.5 x 10 7 differentiated THP-1 macrophages cultured in 20 mL RPMI 1640 / 10% FBS. After 4 h incubation, cell cultures were washed 3× with 37°C PBS to remove extracellular CFP and cultured in RPMI 1640 without FBS for 48h, after which culture supernatant were collected to generate samples for EV and soluble protein analyses. Cultured macrophages were recovered by trypsin digestion and split into samples that were analyzed for viability and employed to generate cell lysates for Western blot analysis of target proteins. . Solid lines denote the mean ± SD of each group. No significant NEI differences were identified between these groups by age or sex (ns: p>0.05 by two-sided Mann-Whitney U-test), and only the EPTB group differed from the non-TB control group, likely due to the limited statistical power of the analysis (ns: p>0.05, or * p<0.05 and ** p<0.01 by two-sided non-parametric Kruskal-Wallis one-way ANOVA with Dunn's post-test). Mean ± SD of three technical replicates per sample, dashed lines indicate the threshold for positive signal determined in corresponding ROC analysis.
Supplementary Fig. 12 | Flow chart for inclusion and exclusion of the DR cohort children and their subgroup categorizations. Confirmed TB cases were defined by positive Mtb culture and/or Xpert results. Children were classified as unconfirmed TB cases if they met the acceptance thresholds for at least two of the following criteria: TB-associated symptoms, a TB-consistent abnormal CXR, had a positive TST result or a known TB exposure, and/or had a positive TB treatment response. Children who lacked any evidence of TB disease or infection, were age-matched to and enrolled from the same neighborhoods as the TB cases. Children enrolled in the non-TB control group who subsequently had positive TST results were excluded and replaced by enrollment of another age-matched control that met the enrollment criteria for this group. Supplementary Fig. 13 | NEI signal in DR cohort children classified into confirmed, unconfirmed, and non-TB groups, as determined by respiratory culture/Xpert or stool Xpert results, TB-related symptoms that met NIH thresholds, TB-suggestive chest X-ray (CXR) findings, close TB contact or TST results. a 3 participants with Unlikely TB but missing CXR, therefore unable to further categorize. b Includes 137 with samples analyzed at baseline; 7 at later visit at time of TB diagnosis and 3 with unlikely TB with first samples available <14 days after enrollment c Assessed during study: Clinical TB diagnosis -initiated TB treatment; No clinical TB diagnosis -did not initiate TB treatment d NIH criteria: TB symptoms (persistent cough (>14 days), fever (>7 days), failure to thrive, lethargy (>7 days)), CXR consistent with TB, or TB exposure within last 2 years or TST ≥5mm. Failure to thrive=wasted (WHZ<-2 or MUAC<12.5) or underweight (WAZ<-2) at enrollment.

Classification
Definition Unlikely TB death Child does not meet criteria for unconfirmed TB prior to death and has an alternate etiology of death (presumed or confirmed). For cases in which the alternate etiology is presumed but the expert panel is unable to definitively rule out intrathoracic TB (respiratory symptoms and/or diagnosis of pneumonia), the death is determined to be unlikely related to TB if the alternate etiology is considered more likely than TB. Possible TB death Child meets classification criteria for unconfirmed TB prior to death.
If the child does not meet unconfirmed TB classification criteria, their death is considered as possibly related to TB if their clinical presentation is compatible with intrathoracic TB (respiratory symptoms and/or diagnosis of pneumonia) and no alternate diagnosis is more likely. Likely TB death Child meets classification criteria for confirmed TB.